Progressive loss of beta cell function is the hallmark of Type 1 diabetes (T1D). Elements of both metabolic and autoimmune markers are known in patients with T1D, although features of early, pre-clinical disease are less well understood. Studies proposed herein combine the unique and complementary expertise of three laboratories with the goal of defining specific parameters during the dysfunction and eventual demise of beta cells, though those cells are asymptomatic in subjects at-risk for T1D. Presented below, our investigators have identified specific biomarkers of beta cells in three important areas, including epigenetics, in the modification of cellular proteins under conditions of stress, and in the identification of unique phenotypes of beta cells that undergo dedifferentiation. There is an important and natural synergy between these three areas of investigation that will better define the changes in beta cells from the nucleus to the cell surface over the course of disease. In all studies, human beta cells will be examined ex vivo in parallel with murine systems to model the metabolic and inflammatory stress of T1D. First, we have identified increased levels of unmethylated insulin DNA (INS), which is of beta cell origin, in the serum of human patients indicating beta cell killing during the preclinical period of the disease. Similar approaches will extend these observations to intracellular genetic methylation events linked to beta cell stress. Second, we have identified specific posttranslational protein modifications (PTMs) in beta cells as well as extracellular products of beta cells in murine models of disease. These observations will now be examined in human islet cells under various conditions of inflammation and dedifferentiation. Finally, we have identified dedifferentiation of beta cells in settings of metabolic stress and postulate that similar changes in the differentiation state occur during development of T1D. The latter observations indicate that beta cells may undergo developmental changes, but not necessarily death, in the development of disease. In our proposed studies, we will evaluate changes in these parameters in a common model system reflecting the immunologic stress that characterizes T1D. We will integrate the findings in order to develop a defined pathway of markers associated with beta cell stress and death. Finally, we will test whether the information we have gathered from our studies can be used as biomarkers using samples obtained from patients with Type 1 diabetes and those at-risk who participated in the DPT-1. Our proposed studies may lead to new understanding of the biologic changes leading to beta cell death in diabetes and ways in which this information can be used to monitor disease activity and guide the use of therapeutics.